Purification of a rat liver cytosolic sulfotransferase responsible for the conjugation of digitoxigenin.
Previous investigations on the digitoxin metabolism hardly considered the role of the sulfate ester conjugation. Therefore, this study examined whether digitoxin (dt-3) or one of its cleavage products might be sulfated in vitro. It was proven that digitoxigenin (dt-0) is by far the best substrate for the cytosolic sulfotransferases (ST). Digitoxigenin-monodigitoxoside (dt-1) and digitoxigenin-bisdigitoxoside (dt-2) are sulfated in trace amounts whereas dt-3 is not sulfated at all. The purification of the responsible enzyme was performed by liquid chromatography on Q-Sepharose and hydroxyapatite. During the purification procedure this enzymatic activity corresponded exactly to that towards dehydroepiandrosterone (DHEA). The 134-fold purified and gel electrophoretically homogeneous enzyme protein (Mr 33,000) showed a Vmax of 12.5 nmoles dt-0 sulfate/min mg protein and a KM of 37 mumol/L. The purified enzyme conjugated dt-1 and dt-2 in trace amounts only and was inhibited competitively by DHEA. It can be concluded that in the rat a 3 beta-hydroxy-steroid sulfotransferase is responsible for the sulfation of dt-0. The purified enzyme reacts with dt-1, dt-2 and digoxigenin (dg-0) in traces only, a sulfation of dt-3 is not detectable.
Johannes
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Institute for Legal Medicine, University of Hamburg, Federal Republic of Germany.
von Meyerinck
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Schmoldt
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D000818:Animals; D002852:Chromatography, Ion Exchange; D003600:Cytosol / Q000201:enzymology; D004073:Digitoxigenin / Q000378:metabolism*; D004074:Digitoxin / Q000378:metabolism*; D004076:Digoxigenin / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D005260:Female; D007700:Kinetics; D008099:Liver / Q000201:enzymology*; D051381:Rats; D011919:Rats, Inbred Strains; D015238:Sulfotransferases / Q000302:isolation & purification*; D013466:Sulfurtransferases / Q000378:metabolism
D004073:Digitoxigenin; D004074:Digitoxin; D013466:Sulfurtransferases; D015238:Sulfotransferases; C028152:alcohol sulfotransferase; D004076:Digoxigenin